Efficient haploid cell sorting flow cytometer systems

ABSTRACT

A flow cytometer system for sorting haploid cells, specifically sperm cells, may include an intermittingly punctuated radiation emitter, such as a pulsed laser. Embodiments include a beam manipulator and even split radiation beams directed to multiple nozzles. Differentiation of sperm characteristics with increased resolution may efficiently allow differentiated sperm cells to be separated at higher speeds and even into subpopulations having higher purity.

I. FIELD OF THE INVENTION

The present invention relates to a flow system for particle analysis. More specifically, the invention relates to the use of a pulsed laser on a flow system for particle analysis which results in more accurate quantification of measurable properties of individual particles. It may be of particular interest in analyzing populations of very similar particles, at high speeds, allowing more efficient separation of particles into two or more different populations. The invention is particularly useful in the application of separating live X-chromosome bearing and Y-chromosome bearing sperm of all mammals at higher speeds, better purities and with equal or better sperm health outcomes, meaning less damage to sperm. The invention may contribute significant improvements to the economics of sperm sorting.

II. BACKGROUND OF THE INVENTION

Lasers can be used to deliver light to biological or non-biological particles and emission spectra can be used for the analysis of particle characteristics. In some instances, this can be applied such as where a particle is self florescent or self color absorbing, is associated by affinity, avidity, covalent bonds, or otherwise to another molecule which may be colored or fluorescent, may be associated to another molecule which is colored or fluorescent through a specific biological or modeled macromolecular interaction, such as an antibody binding event or a nucleic acid oligomer or poynucleic acid hybridization event, may obtain color or fluorescence such as through an enzymatic synthesis event, an enzymatic attachment or cleavage reaction, enzymatic conversion of a substrate, association of a florescent molecule with a nearby quencher, the reaction of a product in certain local proton (pH) or NADH or NADPH or ATP or free hydride (H—) or bound hydride R—(H—) concentrations, or may gain color or fluorescense by way of a variety of methods to associate emitted or absorbed light (electromagnetic radiation EMR).

Conventional lasers can generate a strong, perhaps intense, source of light. Through coherence properties of the beam such light may travel very long distances, perhaps across reflective mirrors which may change the angle of the light illumination beam, perhaps through prisms or refractive objects or lenses which may split it into two or more beams of equal or differing intensity, or may defocus, perhaps expand, or focus, perhaps concentrate, the beam. Such light may also be affected by filters which may reduce the net energy of the beam. Most lasers also allow the modulation of light intensity, perhaps watts, in the beam by adjustment of an input current from a power supply to the light generating element.

In some applications, conventional lasers used in the analysis and quantification of biological objects can be combined with sensitive light detectors that may be as simple, such as a photographic film or paper, or may be more complex, such as a photomultiplier tube. Often, a light detector may collect only information about a cumulative amount of light, perhaps electromagnetic radiation, EMR, or it may collect and report on the dynamic changes in intensity of light or EMR hitting all of, or portions of, localized regions of, or positions on the detector surface. The light detector may also involve use of a photoelectric coupling device, which may allow the energy of photons absorbed on the EMR by the light detector to be converted to current proportional to the incident light or EMR on the light detector surface. The photoelectric coupling device can even be integrated into an electronic circuit with an amplifier which may increase the signal or create gain such that the fluctuations or perhaps summation of amplified current may be available to an analog or digital logic circuit. Designs may also transmit a signal or data set to a user of a particle analysis instrument and this signal may be proportional to the static, cumulative, or perhaps even dynamic intensity of the light or EMR incident upon the detector.

In certain uses of laser light to analyze biological particles, a detector may measure the change in intensity of the source light after incidence upon a particle(s) being analyzed using a reference beam which takes a path without incidence upon the particle(s). In other uses of laser light, modified or unmodified particles take up a fraction of the illumination light or EMR and may emit light of a different frequency. In many cases, the presence of emission light or EMR of a certain wavelength can be used to identify or to quantify characteristics associated with specific particles, or quantitatively measure the amount or number of the specific biological particles present in the sample or in a specific region of or position in the sample.

In some cases, it can be useful to accurately determine very small differences in the illumination light or emission light from two very similar biological particles (for example an X-chromosome bearing sperm cell versus a Y-chromosome bearing sperm cell). These small differences can be analyzed by way of serial presentation of perhaps 50,000 separate emission events per second in a liquid stream. These can also be thousands of separate emissions from molecules (nucleic acids or proteins as examples) on an array field allowing analysis of genetic, genomic, proteomic, or glycomic libraries.

The traditional type of laser used for the analysis of particles in flow cytometry is a continuous wave (CW) laser. Often this provides a beam of constant intensity. However, in some instances, CW lasers can have particular disadvantages for applications as discussed here. The beam can result in modification or destruction of the sample being observed. For example, with respect to sperm cells, irradiation can result in lower fertility of the sperm cells. Second, in some instances when the laser beam continuously operates, it may be desirable to have a method of interrupting the beam if it is moved from a first location of incidence to a second location of incidence without illumination of intermediate areas.

In U.S. Pat. No. 5,596,401 to Kusuzawa, a pulsed laser may be used for imaging an object, such as a cell, in a flow cytometer. This disclosure may be related to improvements in the capture of images from particles such as coherence lowering modulations. Kusuzawa may teach a use of a continuous wave laser for particle detection and imaging.

In U.S. Pat. No. 5,895,922 and U.S. Patent Application No. 2003/0098421 to Ho, pulsed laser light may be used to illuminate and detect hazardous biological particles dispersed in an airflow stream. The invention may include an ultraviolet laser light and looking for the emission of fluorescence from potentially hazardous biological particles. This disclosure may teach the disadvantages of a laser diode apparatus.

U.S. Pat. No. 6,177,277 to Soini, describes employing a two-photon excitation and/or confocal optimal set-up. The invention may relate to the use of confocal optics to reduce an analysis volume to about 10% of standard analysis volume in a flow cytometer. A pulsed laser may provide short pulses of intense light and may allow the simultaneous absorption of two photons so that a wavelength of illuminating light beam may be longer than an emitted single photon bursts. Background signal may be reduced by use of a filter. The invention may include dual signal processing. The invention as described in Soini, may be beneficial in the analysis of small particles such as erythrocytes and bacterial cells.

In U.S. Pat. No. 6,671,044 to Ortyn, a special analysis optics and equipment may be used in an imaging flow cytometer. The Ortyn disclosure may include analyzing a sex of fetal cells in maternal blood as a method for determining the sex of a child during early pregnancy. Ortyn may indicate that analysis rates from an imaging flow cytometer may be restricted to theoretically maximizing at 500 cells per second.

With respect to particle analysis using laser light, the present invention discloses technology which addresses each of the above-mentioned problems.

For the purposes of this invention, a rapidly pulsed, high intensity pulsed laser may be used. This laser may deliver short pulses of high intensity perhaps lasting about 5-20 picoseconds, followed by intervals between pulses which are 100-1000 times as long as the pulses or about 0.5-20 nanoseconds. The light may have very high peak intensities over the period of about 5-20 picoseconds, and low net energies over the period of about 2-10 microseconds.

Flow cytometry, using a high-speed cell analysis, or high-speed cell analysis and sorting instrument, often relies on a laser light source to illuminate a stream of fluid in which particles are entrained. Particles may be caused to flow by a point of illumination at a rapid rate, often in the range of 500 to 100,000 particles per second. Often the light from the illuminating laser source is of constant intensity. The particles in the analysis stream may be of the same size, and may spend the same amount of time within the area of illumination. The amount of light illuminating each particle in a large population of particles analyzed in series may be identical. A detector may be capable of measuring scattered light, or other types of light emitted by the particle as a result of auto-fluorescence or fluorescence associated with a chemical dye, dye complex, or conjugated dye which may be targeted to one or more types of molecular species contained on or within particles in the population and can determine the identity of a particle and, in some cases, make a measurement of the quantity of a specific molecular target associated with the particle. A specific molecular structure on or even within a particle may be characterized and a quantitative measurement of the amount of associated molecular structure on or even within a particle, may yield information which may be used as a basis for sorting out or separating one type of particle from another.

In a flow cytometer, there may be a very short time duration between the exact moment that a particle is illuminated and the exact moment that a physical manipulation or an electrical condition, may be triggered to elicit separation of a specific particle from a stream containing various particles. An example of a physical manipulation may be charging of a droplet. A specific duration may be called a drop delay period, and the duration may be perhaps as brief as about 100 microseconds or perhaps as long as about 10 milliseconds, and may even be about 1 millisecond. In the case of particle sorting, information may be detected from each particle, computational analysis of the information may be determined, and comparison of the computation to a gating value or perhaps even a selection criteria may be accurately performed within a time period shorter than a duration of the drop delay.

Flow cytometer systems may be useful for measuring an average amount of a specific molecule present on or even within a population of particles. Past systems may not have measured the exact amount of a specific molecule on or even within a population of particles. Factors which can contribute to inaccurate measurements of single particles may include the saturation of a stain or even a conjugate to a particle, variation in the quanta of illumination light, effects from the shape of a particle, and perhaps even electronic noise in the detection apparatus.

An example of a particularly challenging problem is the sorting of X-chromosome bearing and Y-chromosome bearing sperm of mammals at high processing rates and high sorting purities. The population of sperm in most mammals is about 50% X-chromosome bearing and about 50% Y-chromosome bearing. A stain, such as Hoechst 33342, may form complexes with double stranded DNA. A measurement of total Hoechst 33342-DNA complex in each sperm may correlate to the total amount of DNA in each sperm. In general, mammals have larger X chromosomes than Y chromosomes and may have a differential between total DNA contents of X-chromosome bearing over Y-chromosome bearing sperm for various mammals. Such differentials may include: human having about 2.8%; rabbit having about 3.0%; pig having about 3.6%; horse having about 3.7%; cow having about 3.8%; dog having about 3.9%; dolphin having about 4.0%; and sheep having about 4.2%. The differentials may correlate to a relative difference of intensities emitted from a stained sperm being sorted for the purpose of separation of X-chromosome bearing and Y-chromosome bearing sperm.

Significant achievements have been made in developing staining conditions to stain DNA in live sperm with Hoechst 33342, such as, the use of dual orthogonal detection systems to determine sperm orientation, the use of hydrodynamic fluidics to increase the numbers of correctly oriented sperm, the setting of gain on detectors, and even the use of high-speed electronics. In the most efficient use of said achievements, it may be possible in most mammals to simultaneously sort sperm into two populations, X-chromosome bearing, and Y-chromosome bearing, at rates of 2500 per second or higher. It may also be possible to sort sperm to purities of 90% or even higher. There may be, however, a distinct problem in that at rates faster than 2500 per second, the purity of the sample may decrease.

This problem may be understood due to the observation that the co-efficient of variation (CV) in possibly even the best sperm sorting procedures may be between about 0.7%-1.5%, and with poor conditions can even be between about 2%-5%. Since the difference in DNA between X-chromosome bearing sperm and Y-chromosome bearing sperm in mammals may be as low as 2.8% as seen in humans and as high as 7.5% as can be seen in chinchillas, the CV may be lower than the DNA differential in order to achieve a large enough separation of the two populations. Humans have one of the lowest known DNA differentials and may have some of the lowest known maximum purifies in sorting. It may be desirable to improve procedures which can reduce the CV.

A method which has been shown to improve the CV may be to use higher intensities of laser light illumination. For example, it is known to use continuous wave lasers to sort various sperm species with between about 100-200 milliwatts of laser illumination, and possibly with about 150 milliwatts. It has been observed that doubling or tripling the intensity and increasing the power to about 300-500 mW can improve the CV. An improved CV can be most apparent by analysis of the “split” between two peaks on a histogram. Yet, there may be problems associated with an increase of intensity or perhaps even an increase of power with a continuous wave laser. In the case of analyzing a Hoechst 33342 DNA complex with a continuous wave laser, the light source may be near a UV spectrum and may have some ionizing effect upon the DNA complex. Ionizing may then cause changes to the DNA. Accordingly, sperm sorted with high intensities continuous wave lasers such as 300-500 mW may not be as fertile. Another problem may include the energy that it may take to power a continuous laser to deliver about 150 mW of energy at near UV spectrum. Continuous wave lasers may require 10,000 mW or perhaps even more of power. Since there may already be a large amount of electrical power required to run a continuous wave laser at 150 mW, a much larger amount of power may be required to run a continuous wave laser at higher powers. Furthermore, a tube life of ion lasers may be reduced when operating at higher powers. An additional problem with the use of continuous wave lasers may be that the CV may drop significantly when using lower powers such as between about 20-80 mW.

In embodiments, the present invention provides flow cytometer designs which may incorporate the use of 2 or more flow nozzles, and even as many as dozens of flow nozzles, possibly operated by a single sorting instrument. Fields such as microfluidics, optics, electronics, and even parallel processing may be explored. In other embodiments the present invention includes the use of beam splitters to create multiple light beams. Yet, a major problem facing the development of reliable flow analysis and flow sorting in parallel may be the high intensity of laser light needed for analysis at each nozzle. This problem is particularly relevant for applications which require a very low CV in measurement of identical particles.

There is a need to provide flow systems for the analysis and sorting of particles that require a low CV value, yet may require higher laser light intensities, yet higher intensities may have negative effects on sperm and require higher power. In the search for solutions to the problems in flow systems for the analysis and sorting of particles, the field of pulsed lasers represents a possible solution.

Surprisingly, even though sperm sorted on a high speed flow cytometer may be damaged by UV light between about 300-500 mW, it is now shown in this invention that powers between about 100-500 mW may not be damaging to sperm if they are delivered in pulses. In embodiments, this may include a pulse having a peak intensity possibly as much as 1000 times higher than the intensity of a continuous wave laser. Pulsed lasers may be designed as quasi-continuous wave lasers and may have fast repetition rates such as between about 50-200 Megahertz and even up to 80 Megahertz. In embodiments, pulses may be between about 5-20 picoseconds. Pulsed lasers may be ideal for providing pulsed light to a stream of particles being analyzed in a flow cell or a flow cytometer. Particles analyzed in flow cytometers with event rates possibly between 10,000-100,000 Hertz, and even between 20,000-60,000 Hertz, may be illuminated from a few hundred pulses from a laser having repetition rates near 80,000 Hertz. Each pulse may provide an intense amount of energy.

There may be certain industrial uses of flow cytometers, as preparative instruments, which may be economically limited by the traditional methods of processing. It may be desirable to provide systems which facilitate parallel processing for industries such as those that rapidly process mammalian ejaculates for the production of large numbers of live sperm for insemination, those that process blood samples for the recovery of specified cells such as fetal cells, white blood cells, stem cells, hematopoetic cells from bone marrow, and the like. In an embodiment of the present invention, special forms of pulsed laser light can allow a single laser to illuminate a plurality of nozzles, perhaps even while not reducing the CV of the samples analyzed.

As a result, by the use of special forms of pulsed laser light, further improvements in the speed and sample purity can be seen. These types of lasers may be essential in the design and development of new flow cytometers perhaps having multiple sorting streams as well.

III. SUMMARY OF INVENTION

Accordingly, a broad object of the invention may provide a particle analysis system having a pulsed laser which can be operated at a low power.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which may allow detection of smaller differences in illumination or emission to differentiate a particle characteristic.

Yet another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows differentiated particles to be separated into subpopulations having a higher incidence of the desired characteristic.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows multiple particle differentiation systems to be run simultaneously using a single laser.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which affords greater resolving power than conventional particle analysis systems using a CW laser.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which generates from fluorochromes upon irradiation greater light intensity than conventional particle analysis systems using a CW laser.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows differentiated particles to be separated into subpopulations at a greater rate than conventional particle analysis systems using a CW laser.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows sperm cells of any species of mammal to be differentiated with increased resolution into X-chromosome or Y-chromosome bearing subpopulations. The benefits of this object of the invention may allow differentiation of sperm cells having: less DNA bound fluorochrome, less residence time in staining protocols, greater elapsed storage time prior to sorting, or perhaps even less affinity to stain due to having been frozen prior to staining protocols.

Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows sperm cells to be separated into X-chromosome or Y-chromosome bearing subpopulations having higher purity or separated into X-chromosome or Y-chromosome bearing subpopulations at a greater number per second.

Yet another broad object of the invention can be to provide a miniaturized and parallel flow cytometer which allows a multiple of nozzles sorting in tandem to be positioned on the same apparatus, that may allow increases in the production rate of sorting, by increasing the number of nozzles which are sorting on a single apparatus.

Naturally, further independent objects of the invention are disclosed throughout other areas of the specification.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a block diagram of an embodiment of a particle analysis system invention which includes beam manipulators such as optical elements, splitters, filters, directional or the like.

FIG. 2 provides other embodiments of beam manipulators.

FIG. 3 represents a fluidically connected system according to certain embodiments of the invention.

FIG. 4 is a simplified representation of a representative pulse of radiation that may used in some embodiments.

FIG. 5 provides illustrations of characteristics of a pulsed illumination beam.

FIG. 6 provides illustrations which may differentiate characteristics of a pulsed radiation beam from a conventional continuous wave radiation beam.

FIG. 7 shows an embodiment of the invention representing a sorting process having multiple nozzles.

FIG. 8 is a drawing of a flow sort embodiment of the invention.

FIG. 9 provides an expanded diagram showing various embodiments of a multiple nozzle assembly.

FIG. 10 depicts embodiments of certain time intervals and light energy quantities which may be derived from particular properties of pulsed light.

FIG. 11 is a comparison of a pulsed laser radiation beam and a continuous wave laser radiation beam.

FIG. 12 is a representation of an embodiment for a sensing routine.

FIG. 13 is a representation of a comparison of aggregate data from various trial data.

FIG. 14 provides histograms and bivariant plots of X-chromosome bearing and Y-chromosome bearing subpopulations of sperm cells using a flow sort embodiment of the invention which provides a 20 mW pulsed laser beam incident to the sperm cells analyzed.

FIG. 15 provides histograms and bivariant plots of X-chromosome bearing and Y-chromosome bearing subpopulations of sperm cells using a flow sort embodiment of the invention which provides a 60 mW pulsed laser beam incident to the sperm cells analyzed.

FIG. 16 provides histograms and bivariant plots of X-chromosome bearing and Y-chromosome bearing subpopulations of sperm cells using a flow sort embodiment of the invention which provides a 90 mW pulsed laser beam incident to the sperm cells analyzed.

FIG. 17 provides histograms and bivariant plots of X-chromosome bearing and Y-chromosome bearing subpopulations of sperm cells using a flow sort embodiment of the invention which provides a 130 mW pulsed laser beam incident to the sperm cells analyzed.

FIG. 18 provides histograms and bivariant plots of X-chromosome bearing and Y-chromosome bearing subpopulations of sperm cells using a flow sort embodiment of the invention which provides a 160 mW pulsed laser beam incident to the sperm cells analyzed

FIG. 19 provides histograms of sperm cells analyzed with a flow sort embodiment of the invention compared to a histogram of sperm cells from the same sample analyzed with conventional CW flow sort technology.

V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned earlier, the present invention includes a variety of aspects, which may be combined in different ways. The following descriptions are provided to list elements and describe some of the embodiments of the present invention. These elements are listed with initial embodiments, however it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described systems, techniques, and applications. Further, this description should further be understood to support and encompass descriptions and claims of all the various embodiments, systems, techniques, methods, devices, and applications with any number of the disclosed elements, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application.

Referring primarily to FIG. 1, the present invention provides, in embodiments, a radiation emitter used with particle analysis systems. In some embodiments of the invention, a radiation emitter (12) or even an intermittingly punctuated radiation element may convert electrical current into photons of radiation of a specific wavelength and may generate radiation or even laser light for analysis of non-biological or biological particles. Radiation (11) may enter a chamber or region (22) in which it may be modulated or even modified, such as but not limited to establishing a coherent wave form. Radiation may be modified by a beam manipulator (21) such as optical elements before it may illuminate a particle or even a particle sample(s).

In some embodiments, beam manipulators (21) such as optical elements may be used and may be located along a light path. Beam manipulators may include mirrors, optically reflective or even refractive mirrors, partially mirrored surfaces, deflectors, beam splitters, refractive objects, lenses, filters, prisms, lenses, or the like. Beam manipulators may modify or even modulate a pulsed laser light by focusing, condensing, de-focusing, expanding, splitting, or the like. Radiation may be split into multiple beams of equal or perhaps even unequal intensity. A radiation beam manipulator may influence a radiation beam such as by adapting a beam or changing a beam as a particular situation may be needed.

With respect to some embodiments of the invention, positional control elements (24) can provide positional control over a mirror, lens, prism, filter, or other optically relevant components. A positional control element may influence the angle of reflection or even refraction of a beam, and may even ultimately direct a final position of a pulsed laser light beam on a particle or particle sample(s). A positional control element may be a mechanical device which may move a mirror along a path, or it may even be a ratcheting or even a stepped device which can provide large numbers of predefined angles for an optical element. A positional control element(s) (24) may be used at any point in a pulsed laser and even a pulsed light assembly or apparatus to modify a beam of pulsed laser light. An oscillator may provide a constant vibration in an optical element and may define a frequency and amplitude.

In order to provide splitting of radiation into at least two light beams, the present invention may include a beam splitter. This may subject sperm cells to a reduced power of radiation than a power of radiation with was originally emitted from a laser source such as radiation emitter. Examples of reduced radiation may include splitting a radiation beam in half, in a fourth of said originally emitted power or even in an eighth of the originally emitted power, as may be represented by FIG. 2. Of course, there are many options in which to reduce power and all are intended to be included within the scope of this disclosure. With a pulsed laser, for example producing a 1000 mW output split into 8 equal beams of, approximately 120 mW, it may be possible to have 8 flow cytometry nozzles sorting together from one light source.

As to other embodiments of the invention, independent beams of pulsed laser light may be equal or may even be unequal in intensity. Split beams can be derived from a source beam of pulsed laser light by the action of optical splitters. As shown by FIG. 2, a beam splitter (31) such as a prism can divide a beam of radiation into at least two beams of the same frequency and pulse rate, but may have equal or even unequal intensities. Split beams of light may have intensities which can be each less intense than, or even additively close to an equivalent to the original beam which entered a splitter or prism. A complex beam splitter or prism, which with multifaceted, three dimensional geometries can split a beam of pulsed laser light into more than one, certainly two, possibly dozens or even hundreds of independent beams of pulsed laser light.

Complex beam splitters (34) may include a three dimensional packed array of simple splitters or even prisms which can create combined three dimensional geometries of refraction and reflection and can even split a beam of radiation into more than one, certainly two, possibly dozens or even hundreds of independent beams of pulsed laser light. This may differ from a complex beam splitter in that an array of simple splitters and even prisms may allow individual geometries of each component, refractive index of each component, which may allow a much larger number of options. A rotational beam splitter (35) can be rotated on an axis, potentially at very high speeds, such that an extremely large number of pulsed laser light beams may be created to the point where one can no longer speak of actual beams, but rather of each pulse moving in an independent direction, slightly different that the prior pulse. Beam splitters or optical splitters may be used at any point along beam of pulsed laser light.

Another example of a beam manipulator (21) may include filters can be placed in a beam path to modulate or modify a property of a pulsed laser light, and may even reduce the net energy of a particular beam. A large number of filters may be used in series or even in parallel across many different beams of pulsed laser light.

A radiation emitter (12) or even a intermittingly punctuated radiation emitter may be embedded in an integrated fashion into a particle analysis or particle separation system. Alternately, with respect to some embodiments of the invention, a pulsed laser source may be independent and splitters, as described above, may be used to split an original beam of light to provide illumination light to numerous independently operating particle analysis components.

It is understood that a fundamental unit of illumination may be one single pulse of a laser light, and that each pulse or laser light may be split numerous times. Through splitting, filtration or even both, a net energy of any given pulse illuminating a biological object or sample may be perhaps as small as a single photon of light or perhaps as large as an original pulse energy emitted from a laser unit.

It is also understood that the use of splitters, which can divide laser light beams into two or more beams, may increase the number of light beams that can illuminate particles, such as biological objects. In embodiments, a number of pulsed light beams per second that can be directed toward particles can be multiplied with a splitter. It is also understood that the pulses per second may be altered to a desired number of pulses per second, timing of the pulse, and even position the pulse. A pulsed light may be distributed by an apparatus to possibly millions of particles or particle sample(s) which may be located in different positions. Through the use of harmonically synchronized oscillations, rotations, and even geometries, many pulses per second may be delivered to the same particle, sample, or even biological objects. It is particularly understood that, in embodiments, systems may be established with recurring illumination events.

An embodiment of the present invention may include a system fluidicly connected system, such as a flow cytometer system, as seen in FIG. 3. This may be representative of a multiple number of flow cytometry systems that are linked as one system.

Radiation emitters and even intermittingly punctuated radiation emitters, as described in more detail below, may provide one, two, three or perhaps even more radiation beams having specific frequencies, wavelengths, intensities, and even watts to illuminate the type of particles to be analyzed. An intermittingly punctuated radiation emitter may multiply subject radiation to, for example, sperm cells for a first amount of time and may multiply terminate radiation of sperm cells for a second amount of time (41). This may be represented by FIG. 4.

In embodiments, a first amount of time (40) may include an amount of time radiation occurs and this time may be between about 5 to about 20 picoseconds. A second amount of time (41) may be a radiation off time and may be between about 0.5 to about 20 nanoseconds. Of course, other amounts of time for each of a first amount and a second amount of time may be used and all are understood to be included within the scope of this invention. A cycle of a first and second time may be understood as a repetition (42). Each repetition may include a time of about 2 to about 10 microseconds, yet the repetition may vary. In embodiments, a repetition rate may include between about 50 to about 200 MHz and may even include a rate up to about 80 MHz. Other repetition rates are possible and all are meant to be included within the scope of this disclosure. In embodiments, a radiation emitter may be a Nd:YAG, Nd:YVO₄, or the like radiation emitters.

FIG. 4 shows parameters taken into consideration when discussing the differences between continuous wave (CW) lasers or even gas and pulsed lasers or even solid state lasers. As shown by FIG. 4, an intermittingly punctuated radiation emitter (56) may emit radiation that may not be continuous, yet be in short regular pulses which may have a duration time or a first amount of time (40). Following a pulse, there may be a dark period or a second amount of time (41) in which no light may be generated. The total elapsed time between two pulses, a repetition rate, may therefore be the duration time in addition to the dark period. A pulse line width and dark period may be similar in length, and it could be postulated that a laser may actually be illuminating a sample for somewhat less than “half of the time”. Alternatively, a pulse line width may be much smaller than a dark period, and thus a sample may be illuminated for only a very small fraction of the time.

Peak energy (36) and a full amount of energy or joules delivered from one pulse of laser light is represented in FIG. 5. Fractional amounts (37) of that energy can be split as described above or put through a neutral density filter. Importantly, one may diminish the amount of light in one pulse used to illuminate a particle by dividing or filtering the beam. For example, a 350 mW beam can be split into 10 equal beams of approximately 35 mW to run 10 independent analysis machines from a single source laser. In practice, the quality of analysis at 35 mW must afford information regarding the characteristics of the particle illuminated, and for commercial applications perhaps afford at least the same amount of information as when particles illuminated with a CW UV laser running at the standard of 150 mW. FIG. 5 may further help in an understanding of the block diagram represented in FIG. 4. Each radiation pulse may be reduced in energy as previously discussed.

An energy density or even watts may be needed to achieve maximum light emission (38) from a particle upon illumination as shown by FIG. 6. It has been contemplated that light input of a continuous wave laser, however, may be so low that a particle may never be fully saturated with illumination (43). An emission light (44) from particles illuminated by a CW laser at a given energy intensity may be constant, as the source light may be constantly refreshing the particle to a certain partial saturation value. By comparison, emission light (39) from the same particle which has been illuminated with pulses may be greater than from a CW laser. Pulses may be short and may have illumination light intensity many orders of magnitude higher than the illumination level of a CW laser. It has also been speculated that a fate of light emission from a particle or particle sample(s) during the dark period may be dependent upon the half-life of emission for the illuminated particle and may even be dependent upon the length of time of the dark period. In the case where a half life may be as long or longer than the dark period, the emission could remain close to maximum during the entire time across all pulses delivered to the sample.

It should also be pointed out that if instead of reducing the input energy by splitting or filtering, one instead uses movement of mirrors and reflectors, one may reduce the number of pulses delivered to a biological sample to as low as one pulse, while retaining the very strong intensity of the pulse. Thus, it is a unique aspect of the invention to provide movement of the full strength pulsed laser beam across a plurality of particles which may for example be entrained in a fluid stream which passes through a flow cytometry nozzle or located on an array or matrix (such as a DNA or protein microarray), or a combination of both, as would be the case of a single laser being oscillated so that it illuminates a small number of flow cytometry nozzles in close proximity.

Now referring primarily to FIG. 8, irradiatable sperm cells may be introduced through a sperm sample injection element (4) which may act to supply irradiatable sperm cells for flow cytometry analysis. Irradiatable sperm cells may be deposited within a nozzle (5) in a manner such that the particles or cells are introduced into a sheath fluid (3). A nozzle may be located in part below an injection point of sperm cells. A sheath fluid (3) may be supplied by a sheath fluid source (46) through a sheath fluid port (2) so that irradiatable sperm cells and sheath fluid may be concurrently fed through a nozzle (5). Accordingly, the present invention may provide establishing a sheath fluid and flowing a sheath fluid into a nozzle, and injecting irradiatable sperm cells into a sheath fluid as shown in FIG. 8.

Further, in embodiments, the present invention may include subjecting irradiatable sperm cells radiation. Radiation may be produced from a radiation emitter (12) as discussed previously. In embodiments the radiation emitter may be a intermittingly punctuated radiation emitter or may even be a continuous wave laser.

In embodiments, the present invention may include multiply subjecting sperm cells to radiation having a wavelength appropriate to activate fluorescence in a sperm cell. The invention may include a fluorescence activation wavelength. Such wavelength may include about 355 nm. Of course, this may include any wavelength that may be needed to activate fluorescence. Such other wavelengths may include 350 nm, 360 nm and other wavelengths and all are meant to be included within the scope of this disclosure.

In embodiments, the present invention may include sufficiently hitting a sperm with radiation to cause an irradiatable sperm to emit fluorescence. This may include providing radiation at certain wavelength, power, energy and the like that is enough to cause an irradiatable sperm to emit fluorescence.

In embodiments, the present invention may provide for exciting irradiatable sperm cells that have been subjected to radiation. When in an excited state, the cells may emit fluorescence. In embodiments, irradiatable sperm cells may be multiply excited with radiation. This may include radiation that is emitted from a intermittingly punctuated radiation emitter.

A pulse of laser light may illuminate particles or even a particle sample(s) at a specific location with an EMR frequency or Hertz, timing such as a clock, intensity or even watts, and even net energy or joules. The particles may absorb the pulsed light, may get excited and may even emit light of the same frequency as that of the pulsed laser light, such as a scatter or may even emit a light of a difference frequency or even fluorescence. The exact nature of the amount of energy absorbed by a particle may be related to the chemical properties of the particle, the chemical properties of any objects attached to or closely associated with the particles, the physical chemical properties of the particle environment, such as biological segregations including membranes, organelles, solutes, pH, temperature, osmolality, colloidal character, or the like, and may even be related to the frequency and intensity of the laser light illuminating the particle. An EMR light emission from a particle, characterized by a wavelength and quantity, can provide highly accurate information about the status of a particle when a pulse of illuminating light is incident. Depending on the nature of the particle and the particle environment, the particle may then emit a florescent light signal, and may do so over a certain period of time defined by a half-life of emission. Typically, a number of pulses of laser light can illuminate a particle or particle sample in a specified period of time, and there can be a corresponding dynamically changing emission of light over the same period, or a time period after illumination.

Emitted fluorescence from each of the sperm cells may be detected with a detection system (23). A detection system may include a fluorescence detector (7) which may be connected to a processing unit (15). While processing the emitted fluorescence, the present invention may include evaluating the emitted signals. Evaluation of emitted fluorescence may include how much fluorescence may be emitted possibly by comparison between the cells or may even possibly be compared to a predetermined number. The present invention may include, in embodiments, selecting an electrical condition to be associated with each of the sperm cells in a sheath fluid flow. An electrical condition may be a charge, voltage or any electrical condition. A drop charge circuit (8) may charge a stream of cells and sheath fluid based upon deduced properties of each of the excited cells. For example, this may be to charge all of the X-chromosome bearing sperm cells with a positive charge, and charging all of the Y-chromosome bearing sperm cells with a negative charge. Of course, while the disclosure focuses primarily upon sperm cells, other particles may be analyzed as discussed in the various embodiments disclosed.

A charged drop may be formed and isolated in a free fall area. A drop may be based upon whether a desired cell does or does not exist within that drop. In this manner the detection system may act to permit a first and second deflection plates (18) to deflect drops based on whether or not they contain the appropriate cell or other item. The deflection plates may be disposed on opposite sides of a free fall area in which a drop may form and the deflection plates may be oppositely charged. As a result, a flow cytometer may act to sort cells by causing them to land in one or more collectors. Accordingly, by sensing some property of the cells or other items, a flow cytometer can discriminate between cells based on a particular characteristic and place them in the appropriate collector. In some embodiments, X-bearing sperm droplets are charged positively and deflected in one direction, and Y-bearing sperm droplets are charged negatively and deflected the other way. A wasted stream which may be unsortable cells may be uncharged and may be collected in collector, an undetected stream into a suction tube or the like.

In this manner, a sheath fluid may form a sheath fluid environment for the sperm cells to be analyzed. Since the various fluids are provided to the flow cytometer at some pressure, they may flow out of nozzle (5) and exit through a nozzle orifice (47). By providing some type of oscillator (6) which may be very precisely controlled through an oscillator control (45), pressure waves may be established within the nozzle and transmitted to the fluids exiting the nozzle at nozzle orifice. Since an oscillator may act upon the sheath fluid, a stream (19) exiting the nozzle orifice (47) can eventually and regularly forms drops (9). Because the particles or cells are surrounded by the fluid stream or sheath fluid environment, the drops (9) may entrain within them individually isolated particles or cells, such as sperm cells with respect to some embodiments of the invention.

In other embodiments, since the drops can entrain particles or cells, the flow cytometer can be used to separate particles, cells, sperm cells or the like based upon particle or cell characteristics. This is accomplished through a particle or cell detection system (23). The particle or cell detection system involves at least some type of detector (7) which responds to the particles or cells contained within fluid stream. The particle or cell sensing system may cause an action depending upon the relative presence or relative absence of a characteristic, such as fluorochrome bound to the particle or cell or the DNA within the cell that may be excited by an irradiation source such as a radiation emitter (12) generating an irradiation beam to which the particle can be responsive. While each type of particle, cell, or the nuclear DNA of sperm cells may be stained with at least one type of fluorochrome different amounts of fluorochrome bind to each individual particle or cell based on the number of binding sites available to the particular type of fluorochrome used. With respect to spermatozoa, the availability of binding sites for Hoechst 33342 stain is dependant upon the amount of DNA contained within each spermatozoa. Because X-chromosome bearing spermatozoa contain more DNA than Y-chromosome bearing spermatozoa, the X-chromosome bearing spermatozoa can bind a greater amount of fluorochrome than Y-chromosome bearing spermatozoa. Thus, by measuring the fluorescence emitted by the bound fluorochrome upon excitation, it is possible to differentiate between X-bearing spermatozoa and Y-bearing spermatozoa.

As a result, the flow cytometer acts to separate the particle or cells by causing them to be directed to one or more collection containers. For example, when the analyzer differentiates sperm cells based upon a sperm cell characteristic, the droplets entraining X-chromosome bearing spermatozoa can be charged positively and thus deflect in one direction, while the droplets entraining Y-chromosome bearing spermatozoa can be charged negatively and thus deflect the other way, and the wasted stream (that is droplets that do not entrain a particle or cell or entrain undesired or unsortable cells) can be left uncharged and thus is collected in an undeflected stream into a suction tube or the like as discussed in U.S. Pat. No. 6,149,867 to Seidel, hereby incorporated by reference herein. Naturally, numerous deflection trajectories can be established and collected simultaneously.

Irradiatable sample cells may include a sample cell that is capable of emitting rays of light upon illumination. This may or may not include having stain molecules attached to a sample particle. Some particles may have features that allow them to emit fluorescence naturally without having to add a stain to them.

Laser light incident upon the particle(s) being analyzed may generate at least one, or perhaps even two, three or more beams of scattered light or emitted light having specific frequencies, wavelengths, intensities and perhaps even watts. All or a portion of the scattered light or even emitted light may be captured by a detector. A detector may include a photomultiplier tube or the like detectors.

A detection system may be used to detecting an amount of emitted fluorescence from each of the sperm cells in a flow cytometry system. A detection system or even a sperm cell fluorescence detector may include a photomultipler tube. In other embodiments a detection system may include an optical lens assembly, a photomultipler tube and even some sort of analysis system such as a computer. An optical lens assembly may collect emitted fluorescence and transport a collected signal to a photomultipler tube. The signal detected by a photomultipler tube may then be analyzed by a computer or the like devices.

A single or perhaps even a multiple digital or analog detector(s) may receive all or even a portion of the scattered light or emission light. Analog or digital processor(s) may convert the signals detected by the detector(s) into analog current or even digital information. The information may accurately represent the identity, frequency, quantity, and even joules of light or EMR received by the detector.

In embodiments, a detector may generate a current or digital signal corresponding to the amount of light quanta hitting the detector. Certain embodiments of the invention, can provide a one dimensional detector which summates all light of the specified wavelength incident upon the detector surface during a specified period of time. The duration or even a total time of detection may be as simple as a fully additive collection or even integration over an entire analysis time of the sample, or it may be a dynamic data set which may record an emission of light from a biological object(s) or sample(s) over a time period. That time period may be as short as the time between two pulses of the pulsed laser light, or it may be as long as many millions of pulses. It is understood that such a detector may become two dimensional when the second dimension of time is considered.

In other embodiments, a two or three dimensional detector may comprise a flat panel, a three dimensional matrix of unit cells or even pixels which can detect an emission light once or more times and can record or report the interaction specific to that individual unit cell. The information relevant to the operator of an apparatus may be a summation or display of the results of many unit cells. This type of detector may include, but is not limited to, photographic film, photographic paper, or even a microchip capable of sending data for imaging on a television screen or computer screen. It is understood that a two dimensional detector may become three dimensional, and three dimensional detector may become dimensional when the additional dimension of time may be considered.

A signal generated by a detector can be processed to provide simple outcomes such as photos. A signal generated by a detector may even be able to allow analysis of many thousands or even millions of biological objects in real time with computer graphics which can give representations to allow a user to modulate or modify an analysis process in real time. In other embodiments, it may be desirable to sort with a magnetic detection.

In embodiments, the present invention may include quantitatively detecting an amount of emitted fluorescence from each of the sperm cells. The quantity of the emitted fluorescence may be detected with a sperm cell fluorescence quantitative detector. Of course this may include other samples. In sperm cells, a X chromosome bearing sperm and a Y chromosome bearing sperm may be distinguished because an X chromosome bearing sperm may emit a different amount of fluorescence than a Y chromosome bearing sperm. In other embodiments, if using other samples besides sperm cells, the present invention may provide distinguishing between a first population of particles and a second population of particles due to a difference in an amount of fluorescence emitted from each population of particles. A distinguishing analysis may be calculated with a detector.

Operating system controlled computer(s) or even graphic user interface controlled computers may use data from an analog or even digital processor(s). A computer may facilitate direct feedback control of a laser and even analytical equipment. A computer may even provide data to support a workstation which may give an operator(s) of an analytical or separation equipment images that may relate to a behavior of the system. This may allow control of the behavior of the system for optimal analysis, quantification, and even separation of a biological object(s) or even a sample(s).

Auxiliary computational, command equipment or even control equipment may provide local network control of analysis, quantification and separation apparatus. Control equipment may communicate in local area networks (LAN) or even wide area networks (WAN) to provide local or perhaps even distant operators capability to initiate, monitor, control, troubleshoot, download data or even instructions, upload data or instructions, terminate, and the like. Control equipment may allow operation of one, two, three or even more apparatus.

Computational subcomponents may correspond to a command and even a control which may be integrated into a pulsed laser design and construction. In some embodiments, computational subcomponents may be independent or even integrated parts of an apparatus and may reside outside a housing of a pulsed laser.

In embodiments, the present invention may provide staining a sperm cell with a fluorescent dye. A stained sperm cell (or in other embodiments, a stained sample) may be stained with fluorochrome and in yet other embodiments, may be stained with Hoechst bisbenzimide H33342 fluorochrome.

In some instances, a large amount of dye may be used to stain a sperm cell. Sperm cells contain deoxyribonucleic acid and deoxyribonucleic acid may have many binding sites that stain (dye) molecules may bind with. Due to the nature of sperm cells, a stained sperm cell may have many molecules of a dye attached to each binding site of a sperm.

In embodiments, the present invention may include staining a sperm sample for a reduced staining time. The staining time may vary due to the type of stain used and even due to the type of sample used, here sperm. Typically, staining sperm with Hoechst 33324 may take about 40 minutes. under other constant conditions. Some examples of a reduced staining time may include the following:

-   -   less than about 40 minutes.     -   less than about 35 minutes;     -   less than about 30 minutes;     -   less than about 25 minutes;     -   less than about 20 minutes;     -   less than about 15 minutes;     -   less than about 10 minutes; and     -   less than about 5 minutes.         Of course, other stain times are certainly possible and all         should be understood as represented within the scope of this         invention.

In embodiments, the present invention provides distinguishing between a X chromosome bearing sperm and a Y chromosome bearing sperm in a flow cytometer system. A X chromosome bearing sperm may emit a different fluorescence from said Y chromosome. For example, a X chromosome bearing sperm may contain more DNA than a Y chromosome bearing sperm, thus a X chromosome bearing sperm may bind to more dye. When illuminated, a X chromosome bearing sperm may emit a greater fluorescence than a Y chromosome bearing sperm. The difference may provide the ability to distinguish the two chromosome bearing sperms.

The present invention may include, in embodiments, minimally staining sperm cells with a fluorescent dye. A minimum sperm stain may include allowing less stain to bind to each sperm. For example, it may take a certain amount of stain to complete attach stain molecules to each binding site of a sperm cell. It may be an efficient option if less amount of stain could be used while maintaining the ability to achieve a desired result, such as the ability to distinguish between two different cells after radiation. In embodiments, the present invention may include providing a percentage of stain. While a percentage of stain may be as low as 10%, other examples may include about 90%, about 80%, about 70% and about 60%. All stain percentages are understood as included within the scope of this disclosure.

A benefit with respect to sorting sperm cells using a pulsed laser can be a reduction in the amount of stain taken up by sperm cells during staining. Because stains or dyes, such as Hoechst 33342, bind with DNA within sperm cells, stain has been considered a factor detrimental to the viability or fertility of sperm cells. Using a pulsed laser flow sort invention, the amount of stain taken up by sperm cells during staining can be reduced by 20% over the amounts used with conventional CW laser cell sorting technology with similar or better resolution of X-chromosome bearing and Y-chromosome bearing subpopulations. In certain sperm cell samples the amount of stain taken up by the sperm cells can be reduced to as little as 60% of the amount used with the same cells sorted by conventional CW cell sorting technology.

Any kind of sample or particle may be used in a flow cytometry system. A sample may include usable cells, reproductive cells, haploid cells, sperm cells, delicate sample, non-biological particles, biological particles, or any kind of cell that can be used with a flow cytometer system. A useable cell may be a cell that can be used for further processing or analysis after completion through a cytometry system. Specifically, in embodiments, this may include providing a viable cell, Reproductive cells may include cells that can be used to reproduce an organism or even a mammal and the like. Haploid cells may include those cells that have a single set of chromosomes, such as sperm cells. A delicate sample may include a sample that is fragile or may even be easily damaged such as reduction in viability. A delicate sample may have increased sensitivity to certain environments such as the type of stain, the sorting process and other environments.

Particles can be non-biological particles such as plastic beads, biological particles such as diploid cells, haploid sperm cells, or the like. It is to be understood that particles are not limited to cells or beads but can also include other non-biological particles, biological particles, and the like. Particles may include, without limitation: the individual binding sites or attachment sites of a molecule on the surface of a cell or other molecule; large molecules (possibly whether on the surface of a cell or within a cell) such as proteins, single stranded DNA, double stranded DNA, mRNA, tRNA, DNA-RNA duplexes, combined protein nucleic acid structures such as a ribosomes, telomerases or the like, DNA or RNA polymerases, samino acid synthetase; the active site of an enzyme such as luciferase, peroxidase, dehydrogenase or even cytochrome oxidase which may require cofactors such as ATP, NADH or NADPH; free or bound hydride (H—); or even any structure biological or non-biological that can be entrained in a fluid stream and made incident to an illumination beam to generate scattered light or even emission light.

In another embodiment that may contribute to efficiency in a flow cytometry system, the present invention may include subjecting sperm cells to low power radiation. While the range of power that may be used with a flow cytometry system may vary, some possibilities for low power may include:

-   -   less than 300 milliwatt;     -   less than 350 milliwatt;     -   less than 200 milliwatt;     -   less than 175 milliwatt;     -   less than 100 milliwatt;     -   less than 88 milliwatt;     -   less than 50 milliwatt; and     -   less than 25 milliwatt.         Again, other powers of radiation are certainly possible and all         should be understood as represented within the scope of this         invention.

In some examples, a Vanguard Laser may be used. The Vanguard Laser is manufactured by Spectra-Physics and can emit 80 million pulses per second (80 MHz). LaserForefront, Spectra-Physics, No. 30 (2001). Each pulse may have line width of about 12 picoseconds, and a repetition rate of about 10 nanoseconds. This may mean that to an approximation, during a single repetition of 10 nanoseconds, the pulsed laser may illuminate a target for about 12 of 10,000 picoseconds or about 0.12% of the total time. In other words, a sample being illuminated by a pulsed laser may be spending approximately 99.88% of the time in the dark. This may also mean that since a pulsed laser may be delivering 350 milliwatts (mW) of total power, during the short 12 picosecond pulse, an average of 280 Watts may be delivered to a particle. This may be 800 times more intense than a light from a continuous wave (CW) laser running at 350 mW. Since reliable sperm sorting can be performed at 150 mW on a standard CW UV laser, which may represent a factor of 653 fold, it could be hypothesized that it may be possible to run a pulsed laser at as low as 150/650 or 0.23 mW and still have light intense enough to illuminate a sperm.

In embodiments, the present invention may include utilizing at least one shared resource to process sperm cells. This may help in efficiency of sperm sorting in flow cytometry systems. A shared resource may include a computer system, a sheath fluid, an integrated multiple nozzle device, and the like. In embodiments, a shared resource may include utilizing one radiation source. Radiation may be split into at least two beams and each beam may be directed toward an nozzle and the sample being sorted. In embodiments, the present invention may include one radiation emitter and a beam splitter or may even include one intermittingly punctuated radiation emitter and a beam splitter. A beam splitter may be any kind of beam splitter as previously discussed.

As discussed above, the use of refractive, or semi-reflective splitters provides multiple beams of pulsed laser light derived from the original source light. These beams may have diminished intensity from the original beam, but may be able to each be used to analyze separate particles or particle sample(s). Also discussed above, each beam may be dedicated to an independent analytical or analytical/separation device (for example, but not limited to, a sperm sorting flow cytometer or cell sorter). In some embodiments of the invention, each light beam corresponding to an independent instrument can be split into two light beams of equal intensity and one light beam made incident upon the particle to be analyzed, and the other light beam can provide a reference beam. By comparing the two beams, the absorption of source light by the particle may be measured. Another unique and important attribute of using a single pulsed laser to supply light to dozens or hundreds or thousands of independent analysis or separating machines may be that the entire complex of instruments served by the single light will be using the same light, and to the extent that all machines are performing identical or highly similar activities, it is possible to use the data from all machines as internal references and standards to each other, and by using computers or both which can give local (LAN) as well as distant (WAN) access to the data, to allow operators or persons at a distance to monitor the performance of each machine in real time.

While multiple nozzles may be integrated into one device, separate flow cytometers having only one nozzle may be lined up so that radiation may be directed to or even split between each nozzle.

In embodiments, the present invention may include flowing at least one sheath fluid and sperm cells into at least two nozzles. By multiplying the number of nozzles operating on a single flow cytometer, the amount (number of particles) analyzed and sorted per unit time may be increased. In the case where the operation of the flow cytometer may be in a production setting representing a saleable product, multiple nozzle may increase the number of units produced in a single shift by a single operator, and thus a reduction in the costs of each unit produced.

By operating a number of nozzles on the same device, a controlling instrumentation used on the flow cytometer and operators of the flow cytometer may use statistical analysis of the performance (operation data) of a multiple of nozzles and may use this data for feedback control of single nozzles within the population of, the nozzles being used. By operating a number of nozzles on the same device, a single light source providing a multiple of beams (one or more for each nozzle) may reside on the same mounting as the nozzles and thereby reduce the complexities of light paths related to nozzles running on individual mountings, which may be independent of the mounting of the primary illumination source. By operating a number of nozzles on the same device illuminated by multiple beams from a single light source providing the capital, operating, parts, service, and maintenance costs from a single laser may be distributed across a multiple of productive sorting nozzles, and, therefore, reduce costs per unit produced which are allocated to the laser operation.

FIG. 7 shows multiple nozzles (5) which can provide charged drops (9). The multiple nozzles and collector (20) may be arranged so that a number of selected containers may be less than a number of nozzles. Selected containers may include containers having collected one specific type of cell, such as all the X chromosome bearing sperm cells. In this figure, the sorted X chromosome sperm cells may be represented by the containers (32). Here there are three selected containers of a selected cell that have been sorted from four nozzles.

In other embodiments, the present invention provides utilizing collected sorted sperm for insemination of female mammals and may even provide for a mammal produced through use of a sorted sperm cells produced with a flow cytometer system according to any of the embodiments as presented in this disclosure.

In other embodiments, the present invention may include individually controlling or even compositely controlling at least two nozzles. Each nozzle may individually adjusted according to a desired function with an individual nozzle control, or a plurality of nozzles may be adjusted compositely with a single nozzle control device that may be connected to each of the nozzles.

Another way to increase efficiency in a flow cytometry system, the present invention includes rapidly sorting said sperm cells. This may be achieved with a rapid sperm sorter or even a rapid particle sample sorter. Sperm may be sorted at any rate. Such possibilities for a sort rate may include:

-   -   greater than 500 cells per second.     -   greater than 1000 cells per second;     -   greater than 1500 cells per second;     -   greater than 2000 cells per second; and     -   greater than 3000 cells per second.         Other sort rates are certainly possible and all should be         understood as represented within the scope of this invention.

In embodiments, the present invention may include a particle sample collector such as a sperm cell collector. A collector may be multiple containers, a combined collector having individual container, or any type of collector. For example and as shown in FIG. 9, a combined collector (63) may include a collector for one type of particle (32), such as X chromosome bearing sperm populations, a container for a second type of particle (33) such as Y chromosome bearing sperm populations, and may even have a third container (64) to collect those drops which may not have been charged.

It may be important in designing illumination beams to consider that the closer the illumination source (laser) may be to an analysis point, the less effect any form of movement such as vibrations may have on the path of the beam. It may be desirable to provide an system which reduces the distance between and location of all nozzles to within a very small distance of each other (for example all within 15 cm), and greatly simplifies and enables the use of multiple beams from a single laser light source.

FIG. 9 shows an exploded diagram of components of a flow cytometry system combined into a parallel construction where a multiple of nozzles may be operated on a single apparatus. Although the diagram depicts six nozzles, it is exemplary, such that it might as easily have only 2 or 3 nozzles, or may have as many as 8 or 10 or 12 or even 24 nozzles side by side.

A multiple of incoming laser beams or radiation (11), which in most embodiments could be equal beams derived by splitting from an original source beam located close to the nozzles, shines onto an analysis point which is defined by the intersection of the beam or radiation (11) with a narrow stream of fluid which emits from the nozzle (5). In some embodiments, the analysis point may be at the focal point (60) of a reflective parabolic dish (61) which may reflect emitted light (62) up to the detection surface (58). Unabsorbed laser light which may not be absorbed may be absorbed by a heat sink, or it may be measured by an additional detector to determine an exact real time intensity of the beam. Each nozzle may be equipped with an oscillator (6) which may provide a force causing the stream emitting from the nozzle tip or orifice (47) to break into droplets at defined frequencies such as in the 10,000-100,000 Hz range. Droplets may be charged, and by action of a magnetic field may be separated. In the case of sorting live mammalian sperm for the presence of X or Y chromosomes, there can be 3 streams of droplets: a stream containing primarily X chromosome bearing sperm, which may by example be collected in one container (32) on one side of a collector (20), a stream containing primarily Y chromosome bearing sperm, which may by example be collected in another container (33) on the other side of a collector (20), and a stream containing sperm which may be dead and which may be collected a third container (64) in the middle of a collector (20). In other embodiments, features such as a detection surface (58), parabolic dishes (61), collectors (20) and in some embodiments nozzles (5) and oscillator(s) (6), may be fabricated into single parts or group subassemblies, which may be sandwiched together to manifest the actual sorting nozzle architecture.

In other embodiments of the present invention, detection surfaces may have diameters (57) similar to the diameter of microliter plate wells, which can be about 5-8 millimeters, and can have distances (59) between two neighboring flow cytometry nozzle tips which are equal to the distance between two wells, which may be about 12-18 mm.

Now, referring primarily to FIG. 10, certain time intervals and light energy quantities which may be derived from the particular properties of light provided by a pulsed laser are shown. The standard lasers used in most flow cytometry and particularly in sperm sorting have been ion tube continuous wave (CW) lasers which emit a fairly constant light flux, pulsed lasers may deliver the same rate of net light. For example, as watts is defined as joules per second, we may consider the period of 1 second. It may be exemplary that for many applications in flow cytometry, as many as 10,000-100,000 individual events may be analyzed in 1 second, so that each event requires illumination energies of approx 1/10,000-1/100,000 joules.

In contrast, the pulsed laser may emit the same net light in regular pulses. In FIG. 15, which represents an arbitrary time axis, each pulse of laser light (68) can emit a certain energy, and have a certain illumination pulse duration. When a pulse may illuminate a particle, a fluorescent emission pulse may occur from that particle which can be represented by an emission curve (65). An emission curve can represents a classic exponential decay function where maximum emission is at the start and the rate of emission (decay) is along a corresponding half life curve. Based on some definition of final decay, for example to the point where emission is 1/1000 of original emission, or about 10 half lives, an emission pulse duration (67) can be established. There may also be a period commencing from the final decay point occurring at the end of the time summated by illumination pulse duration and emission pulse duration (67) and the beginning of the next illumination pulse, which can be defined as the resting period. The total sum of these periods may the period between pulses which may be the interpulse period and is typically the inverse of the frequency of the laser.

Using a detection surface, it may be possible to analyze the light output emitted from a particle emission pulse and specifically measuring the summated total of energy from the emission pulse, which may be an integration of the area (66) under the decay curve (65). This measurement (70) may be stored as an analog electrical charge in a charge storage device such as an appropriate capacitor, of it may be converted to a digital value (70) which can be stored in a digital memory device. Given that the emission pulse occurs as a dynamic emission event, which through a photodiode/amplifier system may be translated in realtime to an electrical current (or voltage differential), measurements of current or voltage at multiple points during the particle emission may allow the derivative of the decay curve to be determined (71). These can be useful values in statistical analysis of multiple identical illumination events of the same particle.

In flow cytometry, which is a broad field in which the instant invention may be used, particles which are being illuminated by a laser are commonly flowing in a fluid stream or a flow cell past a fixed point upon which a laser beam is focused. In FIG. 11, a rate of flow of particles past the point of illumination can be a function of the volume flux of the stream, and the concentration of the particles. An illumination period (72) of time in which the particle is being illuminated may be determined by the flow rate and the size of the particle in the direction of the flow. When the particle may be illuminated by a pulsed laser generating individual emission pulses (73) which can occur in interpulse periods much less than the illumination period (72) of the particle, then a large number of individual emission pulses may be derived from the particle (74). In contrast, when a particle may be illuminated during the same period by a continuous wave (CW) laser, there may be a long emission over the period (75), which can commonly be detected as a peak profile of current over the period. A measurement of the emission from particles illuminated by CW lasers are a single long analog events without natural internal cut points and so either the entire value may be integrated, or the event may be sampled at a discreet multiple of times, or segments of the event are integrated.

In other embodiments, lasers may be used where the illumination pulse duration may be much smaller than an interpulse period which may itself be much shorter than the particle illumination period (72). For example, when the Vanguard Laser is used for the sorting of sperm at approximately 25,000 events per second, the laser which has 80,000,000 illumination pulses per second will deliver approximately 3000 pulses per event, and about 5-10% or 150-300 pulses occur in the particle illumination period (72). Also, the pulse duration is about 10 picoseconds (10−1 1 sec), the interpulse period is about 10 nanoseconds (10−8 seconds), and the pulse emission period is less than 1 nanosecond.

As may be understood from FIG. 12, an illumination pulse may initiating a sensing routine. The instant invention may use a laser pulses as an internal clock to the entire analysis system. Advantages are that each illumination pulse, which is very brief and very strong, can be used to initiate each clock cycle. Within a single clock cycle, a computational subroutine may run which uses the resting period to calculate specific values for each illumination/emission event, and cache the result prior to the initiation of the next clock cycle. An analysis of individual particles could be manifested over a multiple of clock cycles (for example 150-300), such that statistical analysis of all emission events mapping to a single particle may occur, and averaged values related to the measurement of the quantity particle and the position of the particle may be cached. The period between a multiple of events, which may be dominated by periods without emissions, may be used to map the identity and distances and using the momentary gating criteria to effect the sort. Values of operating parameters and results within each sort may be cached for viewing at the 1 minute level, possibly operator status, and graphic or summations for entire sort runs may be generated. In FIG. 17, using the example of sperm being sorted using a Vanguard Laser, the clock cycle may be about 10−8 seconds. Each clock cycle encompasses three periods. The illumination pulse of 10−11 seconds, the emission period of 10−9 seconds, and the resting period of 9×10−9 seconds. Each clock cycle (77) occurs approximately 300 times in each analysis event of 3×10−6 seconds. Time between analysis events averages 2×10−5 seconds. The time between each analysis and the sort (79) is approximately 5×10−2 seconds. Operators will usually want to observe net historical data over the most recent minute (80) and be able to view the progress/history of data over the entire sample from start of sort (81).

There may be many hierarchical layers of data occurring dynamically. At the same time, with a number of nozzles all sorting the same sample at the same time, there are simultaneous events occurring in each nozzle at each hierarchical layer. As it would be labor intensive and inefficient for the operator to control each nozzle, the statistical analysis and algorithmic mapping should allow the operator to view status, history, and averages of all nozzles in aggregate and note only nozzles which are not functioning near the mean of the group. The operator also needs to use commands to change the sorting, which should effect all nozzles at one time.

The data may also be shared between control functions across multiple nozzles and over time to allow the system to make automatic adjustments such as: adjusting optical mirror positions to assure equal laser light in each beam; tracking the performance of each nozzle to make early identification of nozzle occlusion events; tracking the performance of each nozzle to identify differential flow rates and even comparing one or more semen samples with direct comparisons.

All of these various calculations, in real time, can be calibrated very precisely in time, as they may all use the very high frequency laser clock. Thus, in the parallelized flow cytometer, a pulsed laser may serve as an important integration component for all of the data being generated in a multiple of nozzles.

Referring primarily to FIG. 13, since it may be desirable to stain samples just prior to sorting, sorting a sample for a period of time before staining a second sample which may have been sorted and repeating this process several times may create samples which have been stained and sorted at different times, but may be pooled as a single batch. Aggregated data (82) for each sample may be compared across a multiple of nozzles and multiple of sorts (83), for example, from the same ejaculate of a certain bull. Comparisons of the same bull over multiple days (84) and comparisons between various bulls (85) can give a history. The data from this history may reside within the operating system and may be used to assist operators in choosing staining concentrations or times, or to help identify ejaculates which are sorting worse than their normal sorting. Also, if high-throughput resort analysis is available, predicted sex ratios versus. actual sex ratios may be determined, and the aggregated sex ratios may be compared to identify settings and methods which different operators may be using, or to identify operators who are consistently getting lower results. Also, trends in the sorting performance may become visible which dictate special maintenance such as cleaning of optics, replacement of nozzle, mirror assemblies, or the like.

Now referring primarily to FIGS. 14 through 18, embodiments of the invention are shown using a pulsed laser in the context of flow sorting of sperm cells. Various results from experiments run at different powers of radiation beams are shown. The different experiments included 20 mW, 60 mW, 90 mW, 130 mW and 160 mW power and each power was created using neutral density filters. These embodiments can provide high-purity sperm sorting for enrichment of X or Y-chromosome bearing sperm cells which can even be up to 98% in purity.

In yet other embodiments, the present invention may provide collecting at least two populations of sample particles, more specifically, collecting a sorted population of X chromosome bearing sperm and collecting a sorted population of Y chromosome bearing sperm. A collector may be provided to collect each sorted population. Accordingly, the present invention may include a X chromosome bearing sperm collector and a Y chromosome sperm collector. It may be important to sort and collect each population at a high purity. A high purity sorted population of X chromosome bearing sperm and said Y chromosome bearing sperm may include a percentage of purity. Of course, any percentage of purity may exist and some examples may include:

-   -   greater than 85% purity;     -   greater than 90% purity;     -   greater than 95% purity;     -   greater than 96% purity; and     -   greater than 98% purity.         Other percentages of purity are certainly possible and all         should be understood as represented within the scope of this         invention.

Typical pulsed lasers having characteristics similar to that set out by Table 1 or Table 2 can be used with the invention.

TABLE 1 Vanguard 150 mW Output Power Average Output Power [W] 0.15 UV Beam Size [mm] 1 Energy per Pulse [J] 1.875E−09 Peak Power [W] 234-375 Power per cm{circumflex over ( )}2 [W/cm{circumflex over ( )}2] 2.98E+04

TABLE 2 Vanguard 350 mW Output Power Average Output Power [W] 0.35 UV Beam Size [mm] 1 Energy per Pulse [J] 4.375E−09 Power per Pulse [W] 546.875 Power per cm{circumflex over ( )}2 [W/cm{circumflex over ( )}2] 6.96E+04

FIGS. 14 through 18 show univariate histograms and a bivariate dot plots from sorting of Hoechst 33342 stained bovine sperm cells. Sperm cells sorted were obtained from the same freshly ejaculated bull sperm diluted to 200×10⁶ sperm cells per mL and incubated in Hoechst 33342 at 34° C. for 45 min.

With respect to the particular histograms and bivariant plots shown by FIGS. 14 through 19, the event rate (illumination of the sperm cells as they pass through the pulsed laser beam) was established at 20,000 events per second. The sort rate (separation of the sperm cells differentiated by analysis) into subpopulations was varied from 850-3500 depending on the power used. The results are also set out by Table 3.

TABLE 3 Pulsed Laser Power Setting Purity X % Purity Y %  20 mW pulsed 96.5 91.0  60 mW pulsed 93.5 85.5  90 mW pulsed 96.0 89.5 130 mW pulsed 96.0 91.0 160 mW pulsed 97.0 93.0

As can be understood from Table 3, using a pulsed laser sperm cells can be sorted into high purity X-chromosome bearing and Y-chromosome bearing subpopulations. For each laser power setting between 20 mW and 160 mW sorted subpopulations had a purity of up to 97.0% of the correct sex type.

Now referring primarily to FIG. 19, histograms compare the resolution of the same sample of sperm cells using conventional CW flow sorting technology and with an embodiment of the flow sorting invention utilizing a pulsed laser beam. Importantly, pulsed laser illumination of stained sperm cells provides superior resolution of X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells. This is true even when the pulsed laser beam has a power significantly lower than the compared to conventional CW flow sorter technology. Even when the pulsed power is 130 mW, 70 mW or even 20/150 of the power used in the compared to CW flow sorter technology. The histograms of the pulsed laser experiments show a cleaner separation of the two peaks as compared to the continuous wave experiments.

In embodiments, the present invention may include providing a high resolution of a sorted population of sperm cells. A higher resolution may be indicative of the purity of a sorted population. While many different resolution values may exist, some examples of high resolutions may include:

-   -   greater than 7.0;     -   greater than 7.5;     -   greater than 8.0;     -   greater than 8.5;     -   greater than 9.0; and     -   greater than 9.2.         Of course, other resolution values may exist and all are to be         understood as represented within the scope of the invention.

As discussed above, lower laser power analysis of particles, and in this particular embodiment of the invention sperm cells, resolves the long standing problem of having to have a dedicated laser source for each flow sorter. By reducing the laser power required, even without achieving any other benefit, multiple flow cytometers, flow sorters, or cell sorters can be operated using a single laser source. For example, when sorting is accomplished at about 20 mW, a single 350 mW pulsed laser can be used to provide illumination light for as many as 18 separately functioning flow cytometers or flow sorters used to separate sperm cells on the basis of bearing an X or Y chromosome.

Again referring to FIG. 19, another important benefit provided by a pulsed laser invention in the context of sorting sperm cells can be increased resolution of X-chromosome bearing and Y-chromosome bearing subpopulations even when sperm cell samples, such as those used in this specific example, are stored for long periods of time at about 5° C., such as 18 hours or longer, or frozen and thawed prior to staining and analysis, the resolution of sorted sperm cells can improved with the pulsed laser invention compared to conventional CW laser technology.

In embodiments, the present invention may include sorting sperm cells at a low coincidence rate. Some examples of low coincidence rates may include:

-   -   less than 4400;     -   less than 4000;     -   less than 3700; and     -   less than 3600.         Again, other low coincidence rates are certainly possible and         all should be understood as represented within the scope of this         invention.

The present invention may include, in embodiments, collecting sorted populations of sperm cells at a high collection rate. A high collection may increase productivity and even efficiency. Some examples of high collection rates may include:

-   -   greater than 2400 sperm per second;     -   greater than 2600 sperm per second;     -   greater than 2900 sperm per second;     -   greater than 3000 sperm per second; and     -   greater than 3100 sperm per second.         Other collection rates are possible and all are meant to be         included within the scope of this invention.

In yet other embodiments, the present invention may include detecting sperm cells at an event rate of between about 10,000 to about 60,000 sperm cells per second. Of course, an event rate may be greater than 10,000 or even lower than 60,000 cells per second.

A benefit with respect to sorting sperm cells using the pulsed laser invention can be higher sorting speeds. When resolution of a particular sample is increased, the sort rate of subpopulations of sperm cells to a given purity can be increased.

Another benefit with respect to sorting sperm cells using the pulsed laser invention can be a higher purity sort. When resolution of a particular sample is increased, the purity of the subpopulations of sperm cells can be increased.

A pulsed laser invention may be understood to have application with respect to any particular particle characteristic which may be differentiated by change of illumination intensity or by detectable light emission upon illumination with a pulsed light beam. While the applicant has provided specific examples of differentiating the amount of DNA within a cell using the invention, it to be understood that these examples are illustrative of how to make and use the invention with regard to the wide variety of non-biological and biological particles, including, but not limited to, viral particles, polyploid cells, diploid cells, haploid cells (such as sperm cells obtained from any species of mammal such as any type or kind of bovine, ovine, porcine, or equine sperm cells; or sperm cells obtained from any type or kind of elk, deer, oxen, buffalo, goats, camels, rabbits, or lama; or sperm cells obtained from any marine mammal such as whales or dolphins; or sperm cells obtained from any rare or endangered species of mammal; or sperm cells obtained from a zoological species of mammal; or sperm cells obtained from a rare or prize individual of a species of mammal; or sperm cells obtained from an individual of a species of mammal that used to produce dairy or meat products. In embodiments, sperm cells may include any type of sperm cells such as but not limited to, mammals, bovine sperm cells, equine sperm cells, porcine sperm cells, ovine sperm cells, camelid sperm cells, ruminant sperm cells, canine sperm cells and the like.

It is understood that the present invention may exist in unique advantages when combined with other aspects of the various references incorporated.

It is also to be understood that these specific examples provided are not intended to limit the variety of applications to which the invention may be used, but rather are intended to be illustrative how to make and use the numerous embodiments of the invention for application with analytical devices such as flow cytometers, cell sorters, microarray analyzers, imaging microscopes, or microimaging equipment, which may easily be built to contain two or more, and perhaps thousands or even millions parallel channels for analysis, and in such that each of these separate channels is capable to perform the identical or very similar function to a single flow cytometry sorting nozzle, they may be considered “machines”, and it is understood that even a very small device which could be held in a persons hand, may contain many hundreds or many thousands of such “machines” and only be able to function if the use of illumination light is similar or identical to the inventions described herein.

Example 1

Purified fixed bull sperm heads (also described as bull sperm nuclei), stained in standard conditions with DNA binding stain Hoechst 33342, are used as a performance standard to calibrate a sperm sorting flow cytometer prior to the sorting of live sperm. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz illuminates the sample analysis stream in a flow cytometer operating at standard settings and provides the histogram plot shown in Fig. Ex 1. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to resolve bull sperm nuclei into X-chromosome bearing and Y-chromosome bearing populations using standard conditions.

Example 2

A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz illuminates the sample analysis stream in a flow cytometer operating at standard settings sorting said sperm and provides the histogram plot shown in Fig Ex 2. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to resolve live sperm into X-chromosome bearing and Y-chromosome bearing populations under standard conditions.

The above sample is sorted for collection of X-chromosome bearing sperm, and the sort collection rate is 3800 live X-chromosome bearing sperm second. A resort analysis of the sample prepared in said manner measures the purity of said sorted sample to be 95%. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to enrich the content of a sperm population from one in which approximately 50% of the sperm are X-chromosome bearing sperm, to one in which 95% of the sperm are X-chromosome bearing sperm.

Said sorted sperm above are further processed by standard methods for packaging into artificial insemination straws, are cryopreserved by the standard freezing method, and are thawed for analysis of motility of the sperm. Percent motility at various points in the procedure is determined to be: After stain 80%, after sorting 70%, after cooling 65%, after freezing and thawing at 0 minutes 45%, 30 minutes after thawing 45%, 120 minutes after thawing 35%. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to enrich live stained sperm samples which appear normal in respect to the sperm motility.

Example 3

A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitters and neutral density filters, in five separate conditions, to provide illumination energy beam levels of 160 mW (53% of beam power), 130 mW (43% of beam power), 90 mW (30% of beam power), 60 mW (20% of beam power), and 20 mW (6.6% of beam power), respectively, to illuminate the sample analysis stream of a sperm sorting flow cytometer operating at standard settings sorting said stained sperm and providing the 5 histogram plots shown in Fig Ex 3. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to clearly resolve live sperm with energies as low as 60 mW (20% of the beam).

The above samples are sorted at each of the 5 beam energy settings for collection of X-chromosome bearing sperm and Y-chromosome bearing sperm in separate fractions, and the sort collection rate is 850-3500 X-chromosome bearing or Y-chromosome bearing sperm second, depending on the power used, with lower powers associated with lower sort collection rates. A resort analysis of the samples prepared in said manner measures the purity of said sorted samples as shown in Table Ex 3. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser delivering beam energies in the range of 20 mW to 160 mW is consistently able to enrich the content of a sperm population from one in which approximately 50% of the sperm are X-chromosome bearing sperm, to one in which 95% or higher of the sperm are X-chromosome bearing sperm, and simultaneously to one in which 90% or higher of the sperm are Y-chromosome bearing sperm.

TABLE 4 Beam Energy (Pulsed) % Purity - X % Purity - Y  20 mW 96.5 91.0  60 mW 93.5 85.5  90 mW 96.0 89.5 130 mW 96.0 91.0 160 mW 97.0 93.0

Example 4

A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitters and neutral density filters, in two separate conditions, to provide illumination energy beam levels of 130 mW (43% of beam power) and 70 mW (23% of beam power), respectively, to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm. As comparison, the same sample is analyzed on two identical but different flow cytometers operating at standard settings and equipped with a CW (continuous wave) lasers delivering 150 mW in both cases. This demonstrates that even with lower beam energies a standard sperm sorting flow cytometer equipped with the pulsed laser provides superior resolution capability when compared to a same standard sperm sorting flow cytometer equipped with a standard CW laser.

Example 5

A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342 with the standard concentration of Hoechst 33342 being defined as 100% level of stain (control). Two additional samples are prepared which are identical except that they are stained with 80% or 60% of the amount of Hoechst 33342 stain, respectively. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 inn and 80 MHz is equipped with beam splitters and neutral density filters, in two separate conditions, to provide illumination energy beam levels of 150 mW (50% of beam power) and 90 mW (30% of beam power), respectively, to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm with 3 different concentrations of stain used. The resolution between X-chromosome bearing and Y-chromosome bearing sperm for these 6 conditions are provided in the 6 histogram plots shown in Fig Ex 5. This demonstrates that lesser amounts of Hoechst 33342 stain may be used to prepare sperm samples for sorting on a standard sperm sorting flow cytometer, if higher pulsed beam energies are also used.

Example 6

Purified fixed bull sperm heads (also described as bull sperm nuclei), stained in standard conditions with DNA binding stain Hoechst 33342, are used as a performance standard to calibrate a sperm sorting flow cytometer prior to the sorting of live sperm. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz and equipped with a beam splitter to provide an illumination energy beam level of 150 mW (50% of beam power) illuminates the sample analysis stream of a flow cytometer operating at standard settings. Said stained nuclei are analyzed at 20,000 events/second (a rate comparable to the rate used in live bull sperm analysis), as well as at 59,000 events/second. The resolution between X-chromosome bearing and Y-chromosome bearing bull sperm nuclei for these 2 event rate conditions are provided in the 2 histogram plots shown in Fig Ex 6. This demonstrates, that for ideal particles such as nuclei standards, the event rates of analysis may be increased as much as 3-fold with only modest loss in the resolution between X-chromosome bearing and Y-chromosome bearing bull sperm nuclei.

Example 7

Samples of live bull sperm from 4 different bulls were stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitters and neutral density filters, in two separate conditions, to provide illumination energy beam levels of 300 mW (100% of beam power) and 150 mW (50% of beam power), respectively, to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm samples. As comparison, the same samples are sorted on an identical but different flow cytometer operating at standard settings and equipped with a CW (continuous wave) laser delivering 150 mW of energy in the illumination beam. The samples are bulk sorted, which means both X-chromosome bearing and Y-chromosome bearing sperm fractions are pooled. The sorted sperm samples are cryopreserved using standard procedures and the percent of post thaw sperm motilities, as well as the percent of live and dead using PI staining with flow cytometry analysis are scored. The averages for all 4 bulls with the 3 different illumination conditions are shown in Table 5. This demonstrates without statistical significance that all three conditions of illumination yield similar numbers of intact viable sperm after sorting.

TABLE 5 % Motility at % Motility at % Live % Live 0 min 90 min at 0 min at 90 min Laser (mW) post thaw post thaw post thaw post thaw CW (150 mW) 50.0 42.5 43.5 40.6 Pulsed (150 mW) 46.3 42.5 40.0 37.9 Pulsed (300 mW) 48.1 36.3 40.3 37.2

Example 8

Samples of live bull sperm from 5 different bulls are stained in standard conditions with DNA binding stain Hoechst 33342, with the standard concentration of Hoechst 33342 being defined as 100% level of stain (control). Two additional samples from the same 5 bulls are prepared which are identical except that they are stained with 80% or 60% of the amount of Hoechst 33342 stain, respectively.

A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitters and neutral density filters, in two separate conditions, to provide illumination energy beam levels of 150 mW (50% of beam power) and 90 mW (30% of beam power), respectively, to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm samples. As comparison, the same samples are sorted on an identical but different flow cytometer operating at standard settings and equipped with a CW (continuous wave) laser delivering 150 mW of energy in the illumination beam.

For the sorting procedures on all these samples, the average values for resolution (higher values are better), the coincidence rates (lower values are better), the sort collection rates (higher values are better) are compared and shown in Table 6. This demonstrates that sorting efficiencies in all conditions tested with the pulsed laser were equal to or better than the sorting efficiencies achieved using the standard CW laser.

TABLE 6 Stain (%)/ Co-incidence Laser (mW) Resolution rate Sort Rate 100/150 pulsed 8.0 3570 3160 100/90 pulsed 9.3 3560 2610  80/150 pulsed 8.2 3600 3160  80/90 pulsed 9.6 3500 2480  60/150 pulsed 8.6 3600 2940  60/90 pulsed 9.8 3520 2450 100/150 CW 7.6 4380 2720

The same samples are bulk sorted, which means both X-chromosome bearing and Y-chromosome bearing sperm fractions are pooled. The sorted sperm samples are cryopreserved using standard procedures and the percent of post thaw motilities, as well as the percent of live/dead using PI staining with flow cytometry analysis are scored. The averages for all 5 bulls with the 7 different stain and illumination conditions are shown in Table 7. This demonstrates that sperm viability and live counts in all conditions tested with the pulsed laser were equal to or better than the sperm viability and live counts achieved using the standard CW laser and standard stain.

TABLE 7 % Motility % Motility at % Live % Live at 0 min 120 min at 30 min at 120 min Stain (%)/Laser (mW) post thaw post thaw post thaw post thaw 100/150 pulsed 43.3 34.0 36.8 32.4 100/90 pulsed 42.8 33.5 35.3 33.2  80/150 pulsed 42.8 33.8 35.1 35.1  80/90 pulsed 42.0 35.3 35.2 29.7  60/150 pulsed 40.8 31.0 35.8 35.1  60/90 pulsed 41.0 32.8 34.4 33.7 100/150 CW 39.8 33.3 33.4 28.6

Example 9

Samples of live bull sperm from 5 different bulls and 2-6 replicates were stained in standard conditions with DNA binding stain Hoechst 33342 (80%) and bulk sorted under standard conditions in a sperm sorting flow cytometer at event rates of 23,000 sperm/second. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitter to provide illumination energy beam level of 150 mW (50% of beam power) to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm samples.

As a control comparison, same samples of live bull sperm from 5 different bulls and 2-6 replicates were stained in standard conditions with DNA binding stain Hoechst 33342 (100%) and these samples were sorted on an identical but different flow cytometer operating at standard settings and equipped with a CW (continuous wave) laser delivering 150 mW of energy in the illumination beam.

Said various sperm samples were used at concentrations of 200,000 sperm/ml or 1 million sperm/ml to inseminate matured bovine oocytes in standard procedures of in-vitro fertilization (IVF). Cleavage and 2 cell rates at 2.75 days post-insemination, blastocyst development rates at 7.75 days post-insemination, total cell numbers in blastocyst and the blastocyst quality at 7.75 days were measured. The average results for 587 oocytes inseminated with sorted sperm prepared from the system equipped with the pulsed laser, and for 558 oocytes inseminated with sorted sperm prepared from the system equipped with the CW laser are shown in Table 8. Note: lower numbers for blastocyst quality are better. This demonstrates that sperm prepared by a standard sperm sorting flow cytometer equipped with the pulsed laser is capable of fertilizing oocytes in standard IVF procedures and exhibits similar cleavage and blastocyst rates, with the mean quality of blastocysts being slightly better when inseminated with sperm sorted using the standard flow cytometer equipped with the pulsed laser.

TABLE 8 % % % quality of cell counts in Laser (mW) Cleaved 2 cell blastocyst blastocyst blastocyst CW (150 mW) 50.7 27.3 6.7 2.7 131.8 Pulsed (150 mW) 49.7 29.5 5.2 2.1 136.2

Example 10

Samples of live bull sperm from 3 different bulls, on multiple days, were stained in standard conditions with DNA binding stain Hoechst 33342 (100%) and bulk sorted under standard conditions in a sperm sorting flow cytometer at event rates of 20-23,000 sperm/second. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with a beam splitter to provide illumination energy beam level of 150 mW (50% of beam power) to the sample analysis stream of a flow cytometer operating at standard settings sorting said stained sperm samples.

As a control comparison, same samples of live bull sperm from the same 3 different bulls on same days, were stained in standard conditions with DNA binding stain Hoechst 33342 (100%) and these samples were sorted on an identical but different flow cytometer operating at standard settings and equipped with a CW (continuous wave) laser delivering 150 mW of energy in the illumination beam.

Said various sperm samples were used in amounts of 2 million sperm per cyropreserved artificial insemination straw containing 0.25 ml of fluid.

Control straws containing 10 million unsorted sperm, and control straws containing 2 million X enriched sperm sorted using a sperm sorting flow cytometer equipped with a the standard CW laser were used.

In a heterospermic analysis, X-fractions from CW laser sorts were mixed in equal sperm numbers with Y-fractions from pulsed laser sorts to create the #1 comparision. Y-fractions from CW laser sorts were mixed in equal sperm numbers with X-fractions from pulsed laser sorts to create the #2 comparison. Identification of sex in fetuses at 60 days was used as a marker to assign the sex outcome, and accordingly, the likely condition (which laser) can be attributed to successful fertilization. The heterospermic method is particularly useful, as all other factors than sorting procedure are internally controlled in each insemination.

Holstein heifers weighing approximately 750 pounds were synchronized using CIDR/Lutalase. Thereafter observed (AM or PM) for standing heat and were inseminated at 12-24 hours after heat observation. Using 2 inseminators, and a single deep uterine insemination treatment, with 5 test groups spaced approximately one month apart, at a single farm, pregnancy rates and sex of fetus were determined at 60 days post insemination using ultrasonograhy. The results shown in Table 9 demonstrate that the sperm sorted with a standard sperm sorting flow cytometer equipped with a pulsed laser give essentially identical pregnancy rates as sperm sorted using a standard sperm sorting flow cytometer equipped with the standard CW laser.

TABLE 9 % Conception Preganancies/ Sperm dose type Rate Inseminations Unsexed control containing 10 million 62.5 55/88 total sperm X-sexed control containing 2 million 56.4 101/179 total sperm Heterospermic #1 containing 2 million 50.0 45/90 total sperm Heterospermic #2 containing 2 million 58.4 52/89 total sperm Pregancies attributed to CW laser 49.50% 48/97 Pregancies attributed to pulsed laser 50.50% 49/97

Example 11

A sample of live dolphin sperm was collected at poolside, shipped via air freight, and stained with Hoechst 33342 approximately 6 hours after collection. The sorting efficiencies for the single stained sample were then tested on two identical Mo Flo SX sperm sorters, in one case equipped with a pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz equipped with a beam splitter to provide illumination energy beam level of 150 mW (50% of beam power), and the second case with an Innova 90-6 (CW—continuous wave) laser delivering 150 mW of beam energy. The dolphin ejaculate was stained 3 times, and in each case sorted for approximately 2 hours.

Using the CW laser, with sorter event rates at 30,000/sec an average co-incidence rate of 6430/sec was observed, X-chromosome bearing sperm were collected at an average rate of 3450/sec and a total of 72 million sperm were collected in 7 hours for an average recovery of 10.3 million sperm per hour.

Using the pulsed laser, with sorter event rates at 30,000/sec an average co-incidence rate of 5400/sec was observed, X-chromosome bearing sperm were collected at an average rate of 3930/sec and a total of 79.5 million sperm were collected in 6.33 hours for an average recovery of 12.6 million sperm per hour.

The recovered sperm from both samples has X purities of >95% and post-thaw motility of >50%.

Example 12

A sample of live canine sperm was collected from a common dog housed at a kennel and stained about 3 hours later with a non-optimized quantity of Hoechst 33342. The stained sperm were analyzed by a standard sperm sorter equipped with a pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz equipped with a beam splitter to provide illumination energy beam level of 150 mW (50% of beam power). Approximately 43% of the sperm were correctly oriented. From the correctly oriented stained canine sperm, approximately 32% were collected as X-chromosome bearing sperm, and approximately 36% were collected as Y-chromosome bearing sperm. Visual inspection by microscope showed high numbers of canine sperm iii both samples to be motile.

Example 13

The standard CW laser uses a cathode tube which requires an average input of 12 KW of electrical power, and a large volume of cooling water, or a chiller with a load of approximately 15 KW. The pulsed laser (Spectrophysics VNGD350-HMD355 delivering 300 mW of energy at 355 nm and 80 MHz) requires approximately 500 watts (0.5 KW).

The standard CW laser also requires replacement of the cathode tube after approximately 5000 hours of use, at a replacement cost of about $12,000, whereas the VNG pulsed laser is expected to have 30,000+ hours of operation before refurbishment of head element at similar costs.

A commercial operation using the sperm sorting flow cytometers to sort sperm for production of artificial insemination straws, running 24 hours per day, year-round, may be expected to operate lasers for 8,640 hours per year.

Electric utility and water rates in Fort Collins quoted for the year 2004 were used to calculate the operating costs of the standard CW laser, in the first case cooled by utility water and in the second case cooled using electric powered chiller. The pulsed laser requires no cooling. The comparative costs are shown in Table 10. This demonstrates that the pulsed laser has significant benefits in reducing the costs of operation of a sperm sorting flow cytometer.

TABLE 10 CW laser CW laser with with electric Cost component water cooling chiller cooling Pulsed Laser Electrical Charges $4,389 $9,828 $183 Water Charges $6,483 $0 $0 Laser tube or rebuild $20,736 $20,736 $3,456 TOTAL (1 year) $31,608 $30,564 $3,639

As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both sorting techniques as well as devices to accomplish the appropriate sorting system. In this application, the sorting techniques are disclosed as part of the results shown to be achieved by the various devices described and as steps which are inherent to utilization. They are simply the natural result of utilizing the devices as intended and described. In addition, while some devices are disclosed, it should be understood that these not only accomplish certain methods but also can be varied in a number of ways. Importantly, as to all of the foregoing, all of these facets should be understood to be encompassed by this disclosure.

The discussion included in this application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terminology, each element of the device implicitly performs a function. Apparatus claims may not only be included for the device described, but also method or process claims may be included to address the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of the claims included in this or in any subsequent patent application.

It should also be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fall within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) shown, the great variety of implicit alternative embodiments, and the broad methods or processes and the like are encompassed by this disclosure and may be relied upon when drafting the claims for the full patent application. This patent application seeks examination of as broad a base of claims as deemed within the applicant's right and is designed to yield a patent covering numerous aspects of the invention both independently and as an overall system.

Further, each of the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may be expressed by equivalent apparatus terms or method terms—even if only the function or result is the same. Such equivalent broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of a “sorter” should be understood to encompass disclosure of the act of “sorting”—whether explicitly discussed or not—and, conversely, were there effectively disclosure of the act of “sorting”, such a disclosure should be understood to encompass disclosure of a “sorter” and even a “means for sorting” Such changes and alternative terms are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentioned in this application for patent; or patents, publications, or other references mentioned in this application for patent are hereby incorporated by reference. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. Finally, all references listed herein and in the table of references as listed below or other information statement filed with the application are hereby appended and hereby incorporated by reference, however, as to each of the above, to the extent that such information or statements incorporated by reference might be considered inconsistent with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicant(s).

I. U.S. PATENT DOCUMENTS FILING DOCUMENT NO DATE NAME CLASS SUBCLASS DATE 2002/0113965 A1 Aug. 22, 2002 Roche et al. 356 339 Oct. 02, 2001 2002/0141902 A1 Oct. 03, 2002 Ozasa et al. 422 85.09 Mar. 27, 2002 2002/0186375 A1 Dec. 12, 2002 Asbury et al. 356 440 May 01, 2001 2003/0098421 A1 May 29, 2003 Ho 250 458.1 Nov. 27, 2001 2003/0207461 A1 Nov. 06, 2003 Bell et al. 436 172 Nov. 14, 2001 2003/0209059 A1 Nov. 13, 2003 Kawano et al. 73 53.01 Mar. 28, 2003 2004/0005582 A1 Jan. 08, 2004 Shipwash 435 6 Dec. 19, 2002 3,893,766 Jul. 08, 1975 Hogg 356 36 Jun. 14, 1973 4,362,246 Dec. 07, 1982 Adair 209 3.3 Jun. 14, 1980 4,660,971 Apr. 28, 1987 Sage et al. 356 39 May 03, 1984 4,988,619 Jan. 29, 1991 Pinkel 435 30 Nov. 30, 1987 5,088,816 Feb. 18, 1992 Tomioka et. al 356 39 Mar. 06, 1990 5,135,759 Aug. 04, 1992 Johnson 424 561 Apr. 26, 1991 5,315,122 May 24, 1994 Pinsky, et al. 250 461.2 5,371,585 Dec. 06, 1994 Morgan et al. 356 246 Nov. 10, 1992 5,439,362 Aug. 08, 1995 Spaulding 424 185.1 Jul. 25, 1994 5,466,572 Nov. 14, 1995 Sasaki et al. 435 2 Apr. 25, 1994 5,483,469 Jan. 09, 1996 Van den Engh et al. 364 555 Aug. 02, 1993 5,596,401 Jan. 21, 1997 Kusuzawa 356 23 Sep. 14, 1994 5,602,039 Feb. 11, 1997 Van den Engh 436 164 Oct. 14, 1994 5,602,349 Feb. 11, 1997 Van den Engh 73 864.85 Oct. 14, 1994 5,660,997 Aug. 26, 1997 Spaulding 435 7.21 Jun. 07, 1995 5,690,895 Nov. 25, 1997 Matsumoto et al. 422 73 Dec. 06, 1996 5,700,692 Dec. 23, 1997 Sweet 436 50 Sep. 27, 1994 5,726,364 Mar. 10, 1998 Van den Engh 73 864.85 Feb. 10, 1997 5,793,485 Aug. 11, 1998 Gourley 356 318 Jan. 13, 1997 5,895,922 Apr. 20, 1999 Ho 250 491.2 May 23, 1997 5,985,216 Nov. 16, 1999 Rens, et al. 422 73 Jul. 24, 1997 6,149,867 Nov. 21, 2000 Siedel, et al. 422 73 Dec. 31, 1997 6,177,277 B1 Jan. 23, 2001 Soini 436 63 Jan. 03, 1996 6,263,745 Jul. 24, 2001 Buchanan, et al. Dec. 03, 1999 6,357,307 Mar. 19, 2002 Buchanan, et al. 73 865.5 Jul. 20, 2001 6,411,835 B1 Jun. 25, 2002 Modell et al. 600 407 Feb. 02, 1999 6,463,314 B1 Oct. 08, 2002 Haruna 600 407 Feb. 19, 1999 6,528,802 Mar. 04, 2003 Karsten, et al. 250 459.1 Jun. 01, 2001 6,534,308 B1 Mar. 18, 2003 Palsson et al. 435 288.7 Nov. 30, 1999 6,537,829 Mar. 25, 2003 Zarling, et al. 436 514 Dec. 01, 1999 6,577,387 B2 Jun. 10, 2003 Ross, III et al. 356 124 Dec. 29, 2000 6,590,911 B1 Jul. 08, 2003 Spinelli et al 372 22 Jun. 02, 2000 6,604,435 Mar. 13, 2002 Buchanan, et al. Aug. 12, 2003 6,618,679 B2 Sep. 09, 2003 Loehrlein et al. 702 20 Jan. 27, 2001 6,642,018 B1 Nov. 04, 2003 Koller et al. 435 40.5 Mar. 13, 2000 6,667,830 B1 Dec. 23, 2003 Iketaki et al. 359 368 Apr. 09, 1999 6,671,044 B2 Dec. 30, 2003 Ortyn et al. 356 326 Nov. 16, 2001 6,673,095 B2 Jan. 06, 2004 Nordquist 607 89 Feb. 12, 2001

II. FOREIGN PATENT DOCUMENTS DOCUMENT NO DATE COUNTRY EP 0 288 029 20.04.88 Europe WO 96/12171 Apr. 25, 1996 PCT WO 98/34094 Aug. 06, 1998 PCT WO 99/05504 Jul. 24, 1998 PCT WO 99/33956 Jul. 08, 1999 PCT WO 01/40765 Jun. 07, 2001 PCT WO 01/40765 Jul. 06, 2001 PCT WO 01/85913 Nov. 15, 2001 PCT

III. OTHER DOCUMENTS (Including Author, Title, Date, Pertinent Pages, Etc.) Dean, P. N., et al., “Hydrodynamic orientation of spermatozoa heads for flow cytometry”, Biophysical Journal. 23: 7-13, 1978 Elmes, R. S., et al., “Evaluation of the Spectra Physics Vanguard laser as a new UV light source for Flow Cytometry,” Laboratory for Cell Analysis, Comprehensive Cancer Center, University of California, 4 pages     (Date) Fulwyler, M. J. 1977. Hydrodynamic orientation of cells. J Histochem. Cytochem. 25: 781-783. Gurnsey, M. P., and Johnson, L. A., “Recent improvements in efficiency of flow cytometric sorting of X and Y- chromosome bearing sperm of domestic animals: a review”, 1998, New Zealand Society of Animal Protection, three pages. Johnson, L. A., et al., “Enhanced flow cytometric sorting of mammalian X and Y sperm: high speed sorting and orienting No. 77.1e for artificial insemination”, Theriogenology. 49(1): 361. abstr., 1998 Johnson L. A., et al., “Flow cytometry of X- and Y- chromosome bearing sperm for DNA using an improved preparation method and staining with Hoechst 333-42”, Garnete Research 17: 203-212, 1987 Johnson L. A., et al., “Modification of a laser-based flow cytometer for high resolution DNA analysis of mammalian spermatozoa”, Cytometry 7: 266-273, 1986 Johnson, L. A., et al., “Improved flow sorting resolution of X- and Y- chromosome bearing viable sperm separation using dual staining and dead cell gating”, Cytometry 17 (suppl 7): 83, 1994 Johnson, L. A., et al., “Sex Preselection: High Speed Flow Cytometric Sorting of X and Y sperm for Maximum efficiency”, Theriogenology 52: 1323-1341, 1999 Johnson L. A., et al., “Sex preseletion in rabbits: Live births from X and Y sperm separated by DNA and cell sorting”, Bio Reprod 41: 199-203, 1989. Kachel, V., et al., “Uniform Lateral Orientation, Caused by Flow Forces, of Flat Particles in Flow-Through Systems”, The Journal of Histochemistry and Cytochemistry, 1997, Vol. 25, No. 7, pp 774-780. Lightwave Electronics, “Xcyte”, www.LightwaveElectronics.com, 2 pp. “Introducing the Vanguard - 4 Watts of UV from a Quasi-cw, all solid state laser,” LaserForefront, A Monthly Update On The State Of Technologies In The Laser Industry 2001, No. 30 Rens, W., et al., “Improved Flow Cytometric Sorting of X- and Y- chromosome Bearing Sperm: Substantial Increase in Yield of Sexed Semen”, Molecular Reproduction and Development, 1999, pp 50-56. Rens, W., et al., “A Novel Nozzle for More Efficient Sperm Orientation to Improve Sorting Efficiency of X and Y Chromosome-Bearing Sperm”, Technical Notes, Cytometry 33, 1998, pp 476-481. Siedel, G. E. Jr., Herickhoff, L. A., Schenk, J. L., Doyle, S. P. and Green, R. D. 1998. Artificial insemination of heifers with cooled, unfrozen, and sexed semen. 1998. Theriogenology. 49(1): 365 Spectra-Physics Products “FCbar^(tm),” 2 pages, printed Nov. 14, 2002 Spectra-Physics, The Solid State Laser Company “Vanguard 4 Watts of UV from a quasi-cw, all solid state laser.”, 3 pages, printed Nov. 14, 2002 Spectra-Physics, The Solid State Laser Company, “Vanguard 350-HMD 355, www.spectra-physics.com, 3 pp. Spectra-Physics, The Solid State Laser Company, “Vanguard 2000-HM 532, www.spectra-physics.com, 3 pp. Time-Bandwidth ® Products, “GE - 100 - XHP”, www.tbwp.com, 2 pages, January 2002. US National Phase Application Number 09/355,461 filed Sep. 17, 1999 Welch G. R., et al., “Fluidic and optical modifications to a FACS IV for flow sorting of X- and Y- chromosome bearing sperm based on DNA”, Cytometry 17 (suppl. 7): 74, 1994

In drafting any claims at any time whether in this application or in any subsequent application, it should also be understood that the applicant has intended to capture as full and broad a scope of coverage as legally available. To the extent that insubstantial substitutes are made, to the extent that the applicant did not in fact draft any claim so as to literally encompass any particular embodiment, and to the extent otherwise applicable, the applicant should not be understood to have in any way intended to or actually relinquished such coverage as the applicant simply may not have been able to anticipate all eventualities; one skilled in the art, should not be reasonably expected to have drafted a claim that would have literally encompassed such alternative embodiments.

Further, if or when used, the use of the transitional phrase “comprising” is used to maintain the “open-end” claims herein, according to traditional claim interpretation. Thus, unless the context requires otherwise, it should be understood that the term “comprise” or variations such as “comprises” or “comprising”, are intended to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Such terms should be interpreted in their most expansive form so as to afford the applicant the broadest coverage legally permissible. 

1-231. (canceled)
 232. A method of sorting sperm cells comprising the steps of: producing a stream of stained sperm cells and sheath fluid; exciting the stained sperm cells within the stream with a pulsed laser; detecting an emitted fluorescence from each of the stained sperm cells; distinguishing between X chromosome bearing sperm and Y chromosome bearing sperm based on the emitted fluorescence in response to the pulsed laser; isolating sperm cells within droplets; charging the droplets based upon the emitted fluorescence of the sperm cells contained within each droplet; deflecting the charged droplets; and collecting the deflected droplets to form at least one sorted sperm population.
 233. The method of claim 232 wherein the step of producing a stream of stained sperm cells and sheath fluid further comprises the step of injecting stained sperm cells in a sheath fluid within a nozzle.
 234. The method of claim 232 wherein said step of detecting an amount of emitted fluorescence from each of the stained sperm cells comprises the step of detecting an amount of emitted fluorescence from each of the sperm cells with a detection system utilizing a photomultipler tube.
 235. The method of claim 232 wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises subjecting the stained sperm cells to a laser pulse for a pulse duration between about 5 to about 20 picoseconds.
 236. The method of claim 232 wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises providing a pulse resting period between about 0.5 to about 20 nanoseconds.
 237. The method of claim 232 wherein the step of exciting stained sperm cells within the stream with a pulsed laser further comprises providing a repetition rate between 50-200 MHz.
 238. The method of claim 232 wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises providing an average power radiation of less than 300 mW.
 239. The method of claim 232 further comprising the step of staining the sperm cells with a reduced amount of fluorescent dye.
 240. The method of claim 232 wherein the step of staining the sperm cells with a reduced amount of fluorescent dye comprises providing a percentage of stain selected from a group consisting of about 90%, about 80%, about 70% and about 60% of a maximum stain.
 241. The method of claim 232 wherein the sperm cells are selected from a group consisting of non-human mammals, bovine sperm cells, equine sperm cells, porcine sperm cells, ovine sperm cells, camelid sperm cells, ruminant sperm cells, and canine sperm cells.
 242. The method of claim 232 further comprising the step of utilizing at least one shared resource to sort the sperm cells.
 243. The method of claim 242, wherein the step of utilizing at least one shared resource to sort the sperm cells comprises the steps of: utilizing one pulsed laser; splitting the pulsed laser into at least two beams; and directing each of the beams to a different stream of stained sperm cells and sheath fluid.
 244. The method of claim 232 further comprising the step of utilizing said collected sorted sperm for insemination of female non-human mammals.
 245. A non-human mammal produced through use of a sorted sperm cells produced with a flow cytometer system according to claim
 232. 246. A flow cytometry system for sorting sperm comprising: a. a nozzle for producing a stream of stained sperm cells and sheath fluid; b. a pulsed laser for exciting stained sperm cells in the stream; c. a detection system for detecting the emitted fluorescence of stained sperm cells; d. a processing unit for differentiating X chromosome sperm from Y chromosome sperm; e. an oscillator to which the stream is responsive for forming droplets; f. a drop charge circuit to apply a charge to the droplets, wherein the charge applied to each droplet is based on the presence of an X-chromosome or Y chromosome as determined by the processing unit; g. a first and second deflection plate each disposed adjacent to a free fall area in which a droplet forms; and h. at least one collector to collect sorted sperm.
 247. The flow cytometry system for sorting sperm as claimed in claim 246 further comprising a radiation beam manipulator selected from a group consisting mirrors, deflectors, beam splitters, prisms, refractive objects, lenses and filters.
 248. The flow cytometry system for sorting sperm as claimed in claim 247 wherein the detection system comprises a photomultipler tube.
 249. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the stained sperm cells are stained with a fluorescent dye.
 250. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the fluorescent dye comprises Hoechst
 33342. 251. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the pulsed laser comprises an average power less than about 300 mW.
 252. The flow cytometry system for sorting sperm as claimed in claim 246 further comprising a beam splitter.
 253. The flow cytometry system for sorting sperm as claimed in claim 246 further comprising a high purity sorted population of said X chromosome bearing sperm and said Y chromosome bearing sperm.
 254. The flow cytometry system for sorting sperm as claimed in claim 246 wherein said high purity is selected from a group consisting of: greater than 85% purity; greater than 90% purity; greater than 95% purity; greater than 96% purity; and greater than 98% purity.
 255. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the pulsed laser comprises a pulse duration between about 5 to about 20 picoseconds.
 256. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the pulsed laser comprises a pulse resting period between about 0.5 to about 20 nanoseconds.
 257. The flow cytometry system for sorting sperm as claimed in claim 246 the pulsed laser has a repetition rate comprises between about 50 to about 200 MHz.
 258. The flow cytometry system for sorting sperm as claimed in claim 257 wherein the repetition rate comprises up to about 80 MHz.
 259. The flow cytometry system for sorting sperm as claimed in claim 246 wherein the sperm cells are stained with a reduced amount of stain.
 260. The flow cytometry system for sorting sperm as claimed in claim 259 wherein the reduced amount of stain comprises a percentage of stain selected from a group consisting of about 90%, about 80%, about 70% and about 60% of a maximum stain.
 261. The flow cytometry system for sorting sperm as claimed in claim 246 further comprising at least one shared resource, wherein said shared resource comprises a pulsed laser and a beam splitter. 